Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, and are among the most potent toxins known. These toxins are a well-recognized source of food poisoning, often resulting in serious harm or even death of the victims. There are seven structurally related botulinum neurotoxins or serotypes (BoNT/A-G), each of which is composed of a heavy chain ({tilde over ( )}100 KD) and a light chain ({tilde over ( )}50 KD). The heavy chain mediates toxin entry into a target cell through receptor-mediated endocytosis. Once internalized, the light chain is translocated from the endosomal vesicle lumen into the cytosol, and acts as a zinc-dependent protease to cleave proteins that mediate vesicle-target membrane fusion (“substrate proteins”).
These BoNT substrate proteins include plasma membrane protein syntaxin, peripheral membrane protein SNAP-25, and a vesicle membrane protein synaptobrevin (Syb). These proteins are collectively referred to as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Cleavage of SNARE proteins blocks vesicle fusion with plasma membrane and abolishes neurotransmitter release at neuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25 usually reside on the target membrane and are thus referred to as t-SNAREs, while synaptobrevin is found exclusively with synaptic vesicles within the synapse and is called v-SNARE. Together, these three proteins form a complex that is thought to be the minimal machinery to mediate the fusion between vesicle membrane and plasma membrane. BoNT/A, E, and C1 cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), at single but different sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.
Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.
Due to their threat as a source of food poisoning, and as bioterrorism weapons, there is a need to sensitively and speedily detect BoNTs. Currently, the most sensitive method to detect toxins is to perform toxicity assay in mice. This method requires large numbers of mice, is time-consuming, and cannot be used to study toxin catalytic kinetics. A number of amplified immunoassay systems based on using antibodies against toxins have also been developed, but most such systems require complicated and expensive amplification process, and cannot be used to study toxin catalytic activity either. Although HPLC and immunoassay can be used to detect cleaved substrate molecules and measure enzymatic activities of these toxins, those methods are generally time-consuming and complicated, some of them require specialized antibodies, making them inapplicable for large-scale screening. Therefore, there is a need for new and improved methods and compositions for detecting BoNTs.
In the last few years, researchers have started investigating use of FRET assays to detect BoNTs. In FRET assays, two fluorigenic amino acid derivatives are used to replace two native amino acids in a short synthetic peptide (12-35 amino acids) that contain toxin cleavage sites. The fluorescence signal of one amino acid derivative is quenched by another amino acid derivative when they are close to each other in the peptide. This mechanism is called “Förster resonance energy transfer” (FRET). Cleavage of the peptide separates the two amino acid derivatives, such that a decrease in FRET can be detected.
FRET assays have been successfully used for detecting BoNTs. (See e.g., U.S. Pat. App. No. 2004/0191887 to Chapman, filed Oct. 28, 2003, U.S. Pat. App. No. 2006/0134722 to Chapman, filed Dec. 20, 2004, U.S. Pat. No. 7,208,285 to Steward (April 2007), U.S. Pat. No. 7,183,066 to Fernandez-Salas (February 2007), and application US2011/0033866 (publ. Feb 2010),
Although some success has been demonstrated in applying FRET assays to detection of BoNTs, the sensitivity and specificity are still undesirable for many purposes.
In FRET assays for the localization of BoNT substrate proteins, for example, measurements relative to the loss of FRET emission upon cleavage of the peptide can suffer from severe interferences, such that in some cases there is no difference between cells treated with no BoNT versus cells treated with saturating concentrations of BoNT. Therefore, it can be said that methods based on the loss of FRET report BoNT induced changes very poorly and thus its low statistical performance and reproducibility render it a non-reliable methodology. The US application 2009/0191583 to Ester Fernandez-Salas claims a non-FRET BoNT assay using only a single fluorophore. The assays disclosed in this document use cells which are capable of efficient Clostridial toxin uptake and which include a membrane localized Clostridial toxin substrate containing a fluorescent marker.
As an example, a cell useful in this paper can express a SNAP25206-enhanced green fluorescent protein (EGFP) fusion protein that localizes to the plasma membrane. Upon BoNT/A treatment of this cell, cleavage of the membrane localized SNAP25206-EGFP substrate occurs, releasing the EGFP containing fragment into the cytoplasm. Upon excitation of the treated cell with a 484 nM laser, the EGFP is excited and emits light at 510 nM. However, because a portion of the EGFP is now cytoplasmic, a distribution change between the uncleaved, membrane localized SNAP25206-EGFP toxin substrate and the cleaved, cytoplasmic localized EGFP fragment can be observed in BoNT/A treated cells. The assay might therefore work for qualitative analysis, but the presence of EGFP emitting portions in both the membrane and the cytoplasm renders this method undesirable for quantitative analysis.
Thus, improved apparatus, systems and methods are therefore still needed which overcome the drawbacks and the limits of the prior art relative to both FRET and non-FRET assays.